1. Technical Field
The present invention relates to genetic engineering and recombinant DNA technology. More specifically, the present invention provides a process for obtaining natural proteinaceous sweeteners of the thaumatin type, new DNA sequences which have been optimized for expression in filamentous fungi and which code for these proteins, and to the use of these sequences in the transformation of filamentous fungi for the production of thaumatin sweeteners.
2. Background Art
The thaumatins are proteins with a very sweet taste and the capacity to increase the palatability (upgrading or improving other flavors) of food. In industry they are currently extracted from the arils of the fruit of the plant Thaumatoccocus daniellii Benth. Thaumatins can be isolated from these arils in at least five different forms (I, II, III, b and c), which can be separated using ion-exchange chromatography. These forms are all single-chain polypeptides with 207 amino acids and a molecular weight of approximately 22,000 Daltons. Thaumatins I and II, which predominate in the arils and have very similar sequences of amino acids, are much sweeter than saccharose (100,000 times sweeter according to one estimate). Besides being natural products, thaumatins I and II are non-toxic, making them a good substitute for common sweeteners in the animal and human food industries.
Despite its advantages, industrial use of thaumatins of natural plant origin is very limited because of the extreme difficulty involved in obtaining the fruit from which they are extracted. The producing plant, T. daniellii, not only requires a tropical climate and pollination by insects, but it must also be cultivated among other trees and yet 75% of its flowers do not bear fruit.
Attempts have been made to produce thaumatins by genetic engineering in bacteria such as Escherichia coli (see published European patent applications EP 54,330, EP 54,331 and WO 89/06283), Bacillus subtilis and Streptomyces lividans, in yeasts such as Saccharomyces cerevisiae (see WO 87/03007) and Kluveromyces lactis (EP 96,430 and EP 96,910), in the fungus Aspergillus oryzae (Hahm and Batt, Agric. Biol. Chem. 1990, vol. 54, pp. 2513-20), and in transgenic plants such as Solanum tuberosum. Until now, the results of such production efforts have been considered disheartening. Thus, the thaumatin available to industry is very scarce and expensive (cf. M. Witty and W. J. Harvey, "Sensory evaluation of transgenic Solanum tuberosum producing r-thaumatin III", New Zealand Journal of Crop and Horticultural Science, 1990, vol. 18, pp. 77-80, and the articles cited therein).
Accordingly, there has remained a need for economically obtaining industrial amounts of thaumatins.